PROJECT SUMMARY Inherited retinal dystrophies such as retinitis pigmentosa (RP) are a leading cause of blindness for which there is currently no cure. One potential treatment for retinal degenerative diseases is cell- based transplantation therapy, whereby precursor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of photoreceptor precursors into the recipient retina, and the difficulty of obtaining sufficient numbers of photoreceptor precursors for clinical application. To improve protocols for the in vitro culture of such cells, we must have a better understanding of the transcriptional networks that promote specification of photoreceptor precursors. One of the long-term goals of our laboratory is to contribute to these efforts by studying photoreceptor development and regeneration in the zebrafish. The zebrafish is especially useful for studying photoreceptor biology, because its retina contains numerous cone subtypes in addition to rods. Furthermore, unlike mammals, the zebrafish retina is able to regenerate neurons in response to experimental damage. The experiments described in this proposal will define the role of three transcription factors -- Sox, Sox11, and Her9 -- during retinal neurogenesis through the application of gene targeting and molecular genetic approaches. Our specific aims are as follows: Specific Aim I: Determine the role of Sox4 and Sox11 in photoreceptor differentiation. Using newly generated loss-of-function mutants, in this aim, we will 1) determine whether there is a dosage effect of Sox4/11 activity on rod photoreceptor number; 2) use state-of-the-art time-lapse imaging to determine when and where Sox4 expression is required to achieve proper rod photoreceptor development; 3) determine precisely how Sox4/11 regulate Hh and Bmp signaling; and 4) identify the molecular targets of Sox4/11. Specific Aim II: Determine the cause and extent of photoreceptor defects in the zebrafish her9 mutant. In this aim, we will 1) determine the mechanism for photoreceptor defects in her9 mutants; 2) determine whether loss of Her9 impairs vision; 3) identify the upstream signaling pathway(s) that regulate her9 expression in the retina; and 4) determine whether Her9-mediated VEGF expression in the avascular zebrafish retina regulates retinal progenitor cell proliferation and differentiation. Completion of our proposal will bridge important gaps in our understanding of the molecular mechanisms of vertebrate photoreceptor differentiation, and reveal underlying principles relevant to the development of approaches for the treatment of human retinal degenerative disease.